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RNA binding protein isolated from RNA pull-down experiment. Silver stained SDS-polyacrylamide gel containing the proteins pulled down from PK-15 cytoplasmic extracts using agarose beads linked to CSFV ALD 3' UTR. Lane 1 is the protein size marker; lane 2 is proteins from only beads without RNA, lane 3-5 shows 60, 90, and 120 μg of PK-15 cytoplasmic extract used in the pull down reaction. Some of the identity of the proteins are shown with arrows to represent the bands identified by LC/MS/MS.
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Classical swine fever virus (CSFV) is the member of the genus Pestivirus under the family Flaviviridae. The 5' untranslated region (UTR) of CSFV contains the IRES, which is a highly structured element that recruits the translation machinery. The 3' UTR is usually the recognition site of the viral replicase to initiate minus-strand RNA synthesis. Ad...
Citations
... Identification of chemokines in PEVs. LC-MS-MS demonstrated that there were 5,256 proteins in PEVs, and only proteins with >4 distinct peptides and >20% coverage were considered as significant (24,25). GO enrichment analysis with respect to these proteins in PEVs was performed to determine the biological process, cellular component and molecular function. ...
Platelet-derived extracellular vesicles (PEVs), which are generated from the plasma membrane during platelet activation, may be involved in the inflammatory processes of rheumatoid arthritis (RA). The motility of RA fibroblast-like synoviocytes (RA-FLS) plays a key role in the development of synovial inflammation and joint erosion. However, the effects of PEVs on the motility of RA-FLS remain unclear. Thus, the present study aimed to investigate the active contents and potential molecular mechanisms underlying the role of PEVs in regulating the migration and invasion of RA-FLS. The results demonstrated that PEVs contain certain chemokines associated with cell migration and invasion, including C-C motif chemokine ligand 5, C-X-C motif chemokine ligand (CXCL)4 and CXCL7. Furthermore, SB225002, an antagonist of C-X-C motif chemokine receptor 2 (CXCR2; a CXCL7 receptor), partially prevented the migration and invasion of RA-FLS induced by PEVs, suggesting that PEVs may activate a CXCR2-mediated signaling pathway in RA-FLS. In addition, SB225002 antagonized the phosphorylation of IκB and NF-κB in RA-FLS induced by PEVs. Taken together, the results of the present study suggested that PEVs may promote the migration and invasion of RA-FLS by activating the NF-κB pathway mediated by the CXCR2 signaling pathway.
... 3 0 UTRs can affect gene expression by influencing mRNA stability and translation, and relevant cis-acting elements have been reported in human, plant and animal viruses (Nadar et al., 2011;Manzano et al., 2011;Kertesz et al., 2006;Ravet et al., 2012;Barreau et al., 2005;Melanson et al., 2013). Recently, the role of 3 0 UTRs has also been investigated in bacteria. ...
Yersinia pestis, the cause of plague, forms a biofilm in the proventriculus of its flea vector to enhance transmission. Biofilm formation in Y. pestis is regulated by the intracellular levels of cyclic diguanylate (c-di-GMP). In this study, we investigated the role of the 3' untranslated region (3'UTR) in hmsT mRNA, a transcript that encodes a diguanylate cyclase that stimulates biofilm formation in Y. pestis by synthesizing the second messenger c-di-GMP. Deletion of the 3'UTR increased the half-life of hmsT mRNA, thereby upregulating c-di-GMP levels and biofilm formation. Our findings indicate that multiple regulatory sequences might be present in the hmsT 3'UTR that function together to mediate mRNA turnover. We also found that polynucleotide phosphorylase is partially responsible for hmsT 3'UTR-mediated mRNA decay. In addition, the hmsT 3'UTR strongly repressed gene expression at 37°C and 26°C, but affected gene expression only slightly at 21°C. Our findings suggest that the 3'UTR might be involved in precise and rapid regulation of hmsT expression, allowing Y. pestis to fine-tune c-di-GMP synthesis, and consequently regulate biofilm production to adapt to the changing host environment. This article is protected by copyright. All rights reserved.
... For CSFV, enhancement of replication was described to follow induction of autophagic processes (Pei et al., 2014). Further cellular factors interacting with pestiviral proteins are Annexin A2 (interacts with NS5A and is involved in particle production) (Sheng et al., 2015), sphingosine kinase (interacts with NS3 and is thereby inhibited resulting in enhanced virus replication) (Yamane et al., 2009b), adenosine deaminase acting on RNA (ADAR) (interacts with NS4A, overexpression reduces BVDV replication) (Mohamed et al., 2014), and the porcine homologue of human antigen R (HuR) (binds to AU-rich sequences in the 3 0 -NTR of CSFV RNA and might stabilize the RNA) (Nadar et al., 2011). Another group of factors interacts with the core protein, namely the ER-associated degradation pathway protein OS9 and the IQGAP1 protein. ...
Pestiviruses are among the economically most important pathogens of livestock. The biology of these viruses is characterized by unique and interesting features that are both crucial for their success as pathogens and challenging from a scientific point of view. Elucidation of these features at the molecular level has made striking progress during recent years. The analyses revealed that major aspects of pestivirus biology show significant similarity to the biology of human hepatitis C virus (HCV). The detailed molecular analyses conducted for pestiviruses and HCV supported and complemented each other during the last three decades resulting in elucidation of the functions of viral proteins and RNA elements in replication and virus-host interaction. For pestiviruses, the analyses also helped to shed light on the molecular basis of persistent infection, a special strategy these viruses have evolved to be maintained within their host population. The results of these investigations are summarized in this chapter.
© 2015 Elsevier Inc. All rights reserved.
... Many cellular proteins bind to cellular mRNAs that contain AREs, causing either mRNA degradation (e.g., ARE/poly(U)-binding/degradation factor 1 (AUF1)) [48] or protecting the mRNA from degradation (e.g., Hu antigen R (HuR)) [49]. AREs have been found in the non-coding parts of several viral genomes including WNV [50,51]. However, the clear function of these elements in the WNV genome is yet to be determined. ...
West Nile virus (WNV) appeared in the U.S. in 1999 and has since become endemic, with yearly summer epidemics causing tens of thousands of cases of serious disease over the past 14 years. Analysis of WNV strains isolated during the 2006-2007 epidemic seasons demonstrates that a new genetic variant had emerged coincidentally with an intense outbreak in Idaho during 2006. The isolates belonging to the new variant carry a 13 nt deletion, termed ID-Δ13, located at the variable region of the 3'UTR, and are genetically related. The analysis of deletions and insertions in the 3'UTR of two major lineages of WNV revealed the presence of conserved repeats and two indel motifs in the variable region of the 3'UTR. One human and two bird isolates from the Idaho 2006-2007 outbreaks were sequenced using Illumina technology and within-host variability was analyzed. Continued monitoring of new genetic variants is important for public health as WNV continues to evolve.
... The difference is more profound when nonstrand specific annotation was used for analysis (Supplementary Table S4). While HuR's affinity for exons containing Alu elements is not surprising (Brennan and Steitz 2001;Yuan et al. 2010;Nadar et al. 2011;Cherry et al. 2006), this finding raises the possibility of biologic effects from HuR interactions with Alu elements or similar components in the transcriptome playing the role of ''decoys'' (Poliseno et al. 2010;Wang et al. 2009) (see ''Discussion''). ...
The HuR protein regulates the expression of thousands of cellular transcripts by modulating mRNA splicing, trafficking, translation, and stability. Although it serves as a model of RNA-protein interactions, many features of HuR's interactions with RNAs remain unknown. In this report, we deployed the cryogenic RNA immunoprecipitation technique to analyze HuR-interacting RNAs with the Affymetrix all-exon microarray platform. We revealed several thousand novel HuR-interacting RNAs, including hundreds of non-coding RNAs such as natural antisense transcripts from stress responsive loci. To gain insight into the mechanisms of specificity and sensitivity of HuR's interaction with its target RNAs, we searched HuR-interacting RNAs for composite patterns of primary sequence and secondary structure. We provide evidence that secondary structures of 66-75 nucleotides enhance HuR's recognition of its specific RNA targets composed of short primary sequence patterns. We validated thousands of these RNAs by analysis of overlap with recently published findings, including HuR's interaction with RNAs in the pathways of RNA splicing and stability. Finally, we observed a striking enrichment for members of ubiquitin ligase pathways among the HuR-interacting mRNAs, suggesting a new role for HuR in the regulation of protein degradation to mirror its known function in protein translation.
... The cellular proteins such as polypyrimidine tractbinding protein and insulin-like growth factor 2 mRNA-binding protein 1 bind to the 59-and the 39-end of HCV RNA that cause the viral RNA cyclization, and therefore facilitate translation [18,19]. Further, common cellular proteins such as NF45 and RNA helicase A, identified by UV crosslinking assays or RNApulldown assays, were found to associate with the 39 UTRs of CSFV, BVDV, and HCV [18,20,44,45]. These 39 UTR-binding proteins would be expected to be involved in translation/ replication regulation. ...
... Since the template for the translation and replication of a positive-sense RNA virus is the same, the switch between these two processes should be in a tight regulation [46]. The availability of the 39-terminus CGGCCC for the interaction with IRES IIId1 of CSFV in this study could be facilitated by resolving the stem-loop structure in SLI using a cellular protein such as RNA helicase which was reported to interact with the 39 UTR of CSFV [45] . Finally, it is possible that through the longrange 59-39 interaction of CSFV either by base-pairing or with the help of viral [32,33] or host proteins [45], the translation could be well-regulated.Figure S1 Enzymatic probing of the 59 end-labeled LPC 39UTR SL I. A. Summary of the enzymatic structure probing results of SLI. ...
... The availability of the 39-terminus CGGCCC for the interaction with IRES IIId1 of CSFV in this study could be facilitated by resolving the stem-loop structure in SLI using a cellular protein such as RNA helicase which was reported to interact with the 39 UTR of CSFV [45] . Finally, it is possible that through the longrange 59-39 interaction of CSFV either by base-pairing or with the help of viral [32,33] or host proteins [45], the translation could be well-regulated.Figure S1 Enzymatic probing of the 59 end-labeled LPC 39UTR SL I. A. Summary of the enzymatic structure probing results of SLI. B. The RNAs were treated with RNase A (lane A), RNase T1 (lane T1), RNase T2 (lane T2) and RNase V1 (lane V1). ...
The 3' untranslated region (UTR) is usually involved in the switch of the translation and replication for a positive-sense RNA virus. To understand the 3' UTR involved in an internal ribosome entry site (IRES)-mediated translation in Classical swine fever virus (CSFV), we first confirmed the predicted secondary structure (designated as SLI, SLII, SLIII, and SLIV) by enzymatic probing. Using a reporter assay in which the luciferase expression is under the control of CSFV 5' and 3' UTRs, we found that the 3' UTR harbors the positive and negative regulatory elements for translational control. Unlike other stem loops, SLI acts as a repressor for expression of the reporter gene. The negative cis-acting element in SLI is further mapped to the very 3'-end hexamer CGGCCC sequence. Further, the CSFV IRES-mediated translation can be enhanced by the heterologous 3'-ends such as the poly(A) or the 3' UTR of Hepatitis C virus (HCV). Interestingly, such an enhancement was repressed by flanking this hexamer to the end of poly(A) or HCV 3' UTR. After sequence comparison and alignment, we have found that this hexamer sequence could hypothetically base pair with the sequence in the IRES IIId1, the 40 S ribosomal subunit binding site for the translational initiation, located at the 5' UTR. In conclusion, we have found that the 3'-end terminal sequence can play a role in regulating the translation of CSFV.
Human antigen R (HuR) is an RNA-binding protein that regulates the post-transcriptional reaction of its target mRNAs. HuR is a critical factor in cancer development and has been identified as a potential target in many cancer models. It participates in the viral life cycle by binding to viral RNAs. In prior work, we used CRISPR/Cas9 screening to identify HuR as a prospective host factor facilitating Japanese encephalitis virus (JEV) infection. The HuR gene was successfully knocked out in U251 cell lines using the CRISPR/Cas9 gene-editing system, with no significant difference in cell growth between U251-WT and U251-HuR-KO2 cells. Here, we experimentally demonstrate for the first time that the knockout of the HuR gene inhibits the replication ability of JEV in U251 cell lines. These results play an essential role in regulating the replication level of JEV and providing new insights into virus–host interactions and potential antiviral strategies. It also offers a platform for investigating the function of HuR in the life cycle of flaviviruses.
The 3′ untranslated regions (UTRs) of Ebola virus (EBOV) mRNAs are enriched in their AU content and as such represent potential targets for RNA binding proteins targeting AU-rich elements (ARE-BPs). ARE-BPs are known to fine-tune RNA stability and translational activity, particularly of transiently expressed genes. We identified putative AREs within the 3′ UTRs of EBOV nucleoprotein mRNAs and aimed to determine whether these regions are involved in modulating EBOV mRNA post-transcriptional stability as functional targets of ARE-BPs. Using reporter assays designed to study the function of 3′ UTRs in mammalian cells and zebrafish embryos, we show that the tested EBOV 3′ UTRs increase reporter activity, either through enhancing the stability or promoting the translation of reporter mRNA, and that this stabilizing effect is conserved across species. This effect was most pronounced for the nucleoprotein (NP) 3′ UTR. When the EBOV 3′ UTR reporter constructs were co-expressed with tristetraprolin (TTP, ZFP36), a prototypic ARE-BP that is known to mainly destabilize its target mRNAs, only the NP 3′ UTR was responsive as shown by a decrease in reporter gene activity. Co-expression of EBOV NP with TTP led to downregulation of NP protein expression and reduced EBOV minigenome activity, which was dependent on the presence of the NP 3′ UTR, indicating that TTP recognizes cis-acting elements within the NP 3′ UTR. In conclusion, the enrichment of AU residues in EBOV 3′ UTRs makes them possible targets for cellular ARE-BPs, leading to modulation of RNA stability and translational activity.
HuR is an RNA-binding protein implicated in RNA processing, stability, and translation. Previously, we examined protein synthesis in dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We found that the HuR consensus binding element was enriched in transcripts with elevated translation. HuR is expressed in the soma of nociceptors and their axons. Pharmacologic inhibition of HuR with the small molecule CMLD-2 reduced the activity of mouse and human sensory neurons. Peripheral administration of CMLD-2 in the paw or genetic elimination of HuR from sensory neurons diminished behavioral responses associated with NGF- and IL-6-induced allodynia in male and female mice. Genetic disruption of HuR altered the proximity of mRNA decay factors near a key neurotrophic factor (TrkA). Collectively, the data suggest that HuR is required for local control of mRNA stability and reveals a new biological function for a broadly conserved post-transcriptional regulatory factor.
SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in excitability, which may contribute to chronic pain. Noxious cues that promote pain lead to rapid induction of protein synthesis. The underlying mechanisms that confer specificity to mRNA control in nociceptors are unclear. Here, we identify a conserved RNA-binding protein called HuR as a key regulatory factor in sensory neurons. Using a combination of genetics and pharmacology, we demonstrate that HuR is required for signaling in nociceptors. In doing so, we report an important mechanism of mRNA control in sensory neurons that ensures appropriate nociceptive responses to inflammatory mediators.
With their categorical requirement for host ribosomes to translate mRNA, viruses provide a wealth of genetically tractable models to investigate how gene expression is remodeled post-transcriptionally by infection-triggered biological stress. By co-opting and subverting cellular pathways that control mRNA decay, modification, and translation, the global landscape of post-transcriptional processes is swiftly reshaped by virus-encoded factors. Concurrent host cell-intrinsic countermeasures likewise conscript post-transcriptional strategies to mobilize critical innate immune defenses. Here we review strategies and mechanisms that control mRNA decay, modification, and translation in animal virus-infected cells. Besides settling infection outcomes, post-transcriptional gene regulation in virus-infected cells epitomizes fundamental physiological stress responses in health and disease.
